The transcription factor C/EBP? is required for regulation of the balance between differentiation and proliferation during the early stages of myelopoiesis. The importance of this function is demonstrated by the observation that C/EBP? is genetically or functionally inactivated in many types of myeloid leukemia in which the homeostatic coordination between proliferation and differentiation is lost. Moreover, its ectopic expression in myeloid leukemia lines and in primary blast cells from chronic myelogenous leukemia (CML)-blast crisis patients induces granulocytic differentiation and inhibits proliferation; this suggests that restoring the expression of functional C/EBP? or modulating the activity of C/EBP?-regulated effectors required for proliferation and survival of leukemic stem cells may be a novel anti-leukemia therapy. We found that the transcription repressor Gfi-1, which is important for maintaining the quiescence of hematopoietic stem cells, is required for the proliferation inhibitory effects of C/EBP?. Overexpression of Gfi-1 suppresses proliferation and colony formation of BCR/ABL-transformed cells and its inhibitory effects are reversed by restoring the expression of STAT 5 and Mcl-1, two transcriptionally repressed Gfi-1 targets important for proliferation and survival of normal and leukemic stem cells. These findings support the existence of a C/EBP?-Gfi-1-STAT5/Mcl-1 regulatory pathway that could be exploited therapeutically for the elimination of CML stem cells. Thus, two alternative approaches are proposed here to assess the therapeutic potential of perturbing C/EBP?-regulated pathways in CML stem cells: i) the use of C/EBP? itself delivered as a biologically active protein to primitive CML cells; ii) the genetic and pharmacological targeting of C/EBP? -Gfi-1-regulated genes specifically required for the proliferation/survival of CML stem cells.